Journal: Nature Communications

Article Title: A small molecule VDAC ligand inhibits ERAD and induces selective cancer cell death via disruption of calcium homeostasis

doi: 10.1038/s41467-025-67816-z

Figure Lengend Snippet: a ER dislocation can be visualized using NHK-drGFP reporter. Live cell imaging of HeLa cells expressing SP-S11-NHK-HA (ERAD client) and S1-10 (cytoplasmic complementary fragment of GFP). Bortezomib is added to block proteasomal degradation and measure ER dislocation (green). CellMask Deep Red Plasma Membrane Stain and Hoechst 33342 (blue) were used to stain the plasma membrane and nucleus, respectively. NMS-873, a p97 inhibitor, blocks ER dislocation. Imaging was performed using an Opera Phenix High Content Screening System. Scale bar = 100 microns. Images shown are representative of at least three independent experiments. b NMS-873 inhibits ER dislocation and NHK-drGFP fluorescence. A 16-dose titration of NMS-873 was included as a control on six 1536-well assay plates. Fluorescence was measured using an Acumen Laser Scanning Imaging Cytometer, and the IC 50 is indicated. c High-throughput screening of 265 assay plates. NHK-drGP fluorescence was measured for bortezomib (black circles, neutral control, mean ± SD, n = 32 wells per plate) and bortezomib plus NMS-873 (red squares, inhibitory control, mean ± SD, n = 16 wells per plate). d HTS screening funnel describing triage of 96,047 compound screen. Created in BioRender. Henderson, Mark. ( https://BioRender.com/iwzn2wx ) e Six compounds from HTS prioritized for follow-up testing. The compounds were tested at 11-concentrations using two ERAD substrates, NHK and CD3δ. f Effect of compounds on CFTR biogenesis was examined. BHK cells stably expressing HA-∆F508 CFTR were treated with DMSO or different doses of compounds for 24 h followed by processing for immunoblotting. Two distinct CFTR forms were detected: the ER-resident core-glycosylated B form and the fully glycosylated mature C form, representing different stages of CFTR processing and trafficking. Data shown are representative of three independent experiments.

Article Snippet: To monitor assay performance, each plate included a 16 pt titration of the p97 inhibitor, NMS-873 (HY-15713; MedChemExpress), as a reference control.

Techniques: Live Cell Imaging, Expressing, Blocking Assay, Clinical Proteomics, Membrane, Staining, Imaging, High Content Screening, Fluorescence, Titration, Control, Cytometry, High Throughput Screening Assay, Stable Transfection, Western Blot

Journal: Cell Reports

Article Title: The protease SPRTN and SUMOylation coordinate DNA-protein crosslink repair to prevent genome instability

doi: 10.1016/j.celrep.2021.110080

Figure Lengend Snippet:

Article Snippet: Rabbit anti-p97 , Proteintech , Cat#10736-1-AP; RRID: AB_2214635.

Techniques: Virus, Subcloning, Recombinant, Staining, Picogreen Assay, Proliferation Assay, Flow Cytometry, DNA Extraction, Sequencing, Luciferase, Software, Transfection, Modification, Magnetic Beads, Membrane

Journal: Brain : a journal of neurology

Article Title: Strumpellin is a novel valosin-containing protein binding partner linking hereditary spastic paraplegia to protein aggregation diseases.

doi: 10.1093/brain/awq222

Figure Lengend Snippet: Figure 2 (A) Strumpellin (stru) immunoblot analysis of a total protein extract from murine skeletal muscle (left) and mRFPmars immunodetection of mCherry-strumpellin overexpression in 293TN cells (right). (B) Immunoblot analysis of a strumpellin co-immunoprecipitation experiment. CoIP = co-precipitated proteins by the strumpellin antibody; ctrl = beads incubated with bovine serum albumin instead of polyclonal strumpellin antibody; input = soluble lysate of mCherry-strumpellin transfected 293TN cells; RFP = red fluorescent protein; stru = strumpellin; (1) = unspecific signal from the strumpellin antibody; (2) = degradation product of mCherry–strumpellin; (3) = signal from the strumpellin antibody heavy chain. (C) Pull-down experiment using recombinant GST–strumpellin coupled to beads and soluble VCP (GST-stru versus VCP). This experiment confirms a direct interaction between both proteins. As a negative control, beads were coated with GST (GST versus VCP). For illustration purposes individual lines from the original western blot were digitally re-arranged. (1) = strumpellin degradation products.

Article Snippet: Then sections were pre-incubated in 5% bovine serum albumin (0.3% Triton, 0.1 M phosphate-buffered saline, 45 min, room temperature) and incubated either with rabbit polyclonal anti-VCP antibody (dilution 1:500– 1:2000; NB100-1558, Novus Biologicals), rabbit polyclonal anti-strumpellin (dilution 1:50) or rabbit monoclonal anti-huntingtin antibody (1:500; clone EP867Y, Epitomics) diluted in 2% bovine serum albumin, 0.3% Triton, 0.1 M phosphate-buffered saline (48 h, 4 C).

Techniques: Western Blot, Immunodetection, Over Expression, Immunoprecipitation, Incubation, Transfection, Recombinant, Negative Control

Journal: Brain : a journal of neurology

Article Title: Strumpellin is a novel valosin-containing protein binding partner linking hereditary spastic paraplegia to protein aggregation diseases.

doi: 10.1093/brain/awq222

Figure Lengend Snippet: Figure 3 (A) Schematic structure of the human strumpellin gene. The human strumpellin (KIAA0196) locus comprises 68 kbp at position 126.1 Mb in chromosome 8q24.13, adjacent to the SQLE (squalene epoxidase) and NSMCE2 (non-SMC element 2) genes and linked within 1.5 Mbp to annexin A13 (NCBI reference NC_000008.19). It consists of one non-coding plus 28 protein-coding exons and an elevated incidence of phase 2 introns between codon base positions 2 and 3 in all vertebrate species examined. Known non-synonymous polymorphisms (nsSNPs) are indicated as P216 L, L229 R, N471D*, L619 F*, V626 F* (50 exon 16, 141 bp), P730 H and A1049 V identified by HapMap or the 1000 human genomes projects (*based on clinical data). (B) Schematic structure of the human strumpellin protein. Strumpellin consists of an N-terminal domain, a ‘spectrin-like’ repeat domain (five repeats), and a C-terminal domain. No structural similarity to known domains was found for the N-terminal domain. The strumpellin ‘spectrin-like’ repeat is predicted to be structurally similar to Exo70 (protein data bank accession number 2pft) and importin beta-2 (protein data bank accession number 2z5 k). The structure of Exo70 is shown for illustration only; the position of the polyclonal strumpellin antibody epitope (red) and the mutations known to cause hereditary spastic paraplegia (cyan) are highlighted at their approximate positions. Further known non-synonymous polymorphisms are indicated (black). The C-terminal domain of strumpellin has predicted similarity to exportin-5 (protein data bank accession number 3a6p) and importin beta-1 (protein data bank accession number 2 bpt). The structure of exportin-5 is also shown for illustration only. A potential splicing variant is indicated (M149). Prediction of secondary-structure elements of strumpellin as well as structural similarity searches were carried out using PSIPRED (Bryson et al., 2005). Structure-based sequence alignments for the spectrin repeats were generated manually using the structure of the 16th repeat from chicken brain alpha-spectrin [protein data bank accession number 1aj3; (Pascual et al., 1997)] as template. Figure prepared with PyMOL (DeLano, 2002).

Article Snippet: Then sections were pre-incubated in 5% bovine serum albumin (0.3% Triton, 0.1 M phosphate-buffered saline, 45 min, room temperature) and incubated either with rabbit polyclonal anti-VCP antibody (dilution 1:500– 1:2000; NB100-1558, Novus Biologicals), rabbit polyclonal anti-strumpellin (dilution 1:50) or rabbit monoclonal anti-huntingtin antibody (1:500; clone EP867Y, Epitomics) diluted in 2% bovine serum albumin, 0.3% Triton, 0.1 M phosphate-buffered saline (48 h, 4 C).

Techniques: Variant Assay, Sequencing, Generated

Journal: Brain : a journal of neurology

Article Title: Strumpellin is a novel valosin-containing protein binding partner linking hereditary spastic paraplegia to protein aggregation diseases.

doi: 10.1093/brain/awq222

Figure Lengend Snippet: Figure 9 Immunofluorescence analysis of IBMPFD muscle tissue (R155 C VCP mutation) and myofibrillar myopathies due to heterozygous desmin (R350 P), myotilin (S55 F) and B-crystallin (G154 S) mutations. Note that cytoplasmic strumpellin positive pathological protein aggregates (arrows) were present in all cases analysed. In IBMPFD strumpellin labelling was also observed in a VCP positive nucleus (double-arrowhead). Double-arrows denote subsarcolemmal strumpellin and desmin positive structures, whereas arrowheads demonstrate protein aggregates that are exclusively labelled by the strumpellin antibody. mAb = monoclonal antibody; pAb = polyclonal antibody.

Article Snippet: Then sections were pre-incubated in 5% bovine serum albumin (0.3% Triton, 0.1 M phosphate-buffered saline, 45 min, room temperature) and incubated either with rabbit polyclonal anti-VCP antibody (dilution 1:500– 1:2000; NB100-1558, Novus Biologicals), rabbit polyclonal anti-strumpellin (dilution 1:50) or rabbit monoclonal anti-huntingtin antibody (1:500; clone EP867Y, Epitomics) diluted in 2% bovine serum albumin, 0.3% Triton, 0.1 M phosphate-buffered saline (48 h, 4 C).

Techniques: Mutagenesis

Journal: Cell Biology and Toxicology

Article Title: Effects of the LINC00641/miR-323a-3p/EIF4G2 axis on behaviors and brain monoamine neurotransmitters in chronic unpredictable mild stress mice

doi: 10.1007/s10565-025-10015-9

Figure Lengend Snippet: miR-323a-3p can target EIF4G2. A – C EIF4G2 expression level in the hippocampal tissues of mice was measured using RT-qPCR ( A ) and Western blot ( B - C ). For RT-qPCR, total RNA was extracted from hippocampal tissues, reverse transcribed into cDNA, and quantified using specific primers for EIF4G2. For Western blot, protein lysates were separated by SDS-PAGE, transferred to PVDF membranes, and probed with anti-EIF4G2 antibody. D Bioinformatics analysis predicted the binding site of miR-323a-3p and EIF4G2. E Dual luciferase reporter gene assay was performed to validate the regulatory relationship between miR-323a-3p and EIF4G2. HEK293T cells were co-transfected with a luciferase reporter vector containing the WT or Mut EIF4G2 3'UTR and miR-323a-3p mimic or NC. Luciferase activity was measured 48 h post-transfection, demonstrating that miR-323a-3p directly targets EIF4G2. F – H Effect of LINC00641 up-regulation and miR-323a-3p down-regulation on EIF4G2 protein level, as determined by Western blot.; The data in Figure C came from three independent experiments, and in other figures, n = 10 animals in each group. These comparisons were made through Student’s t-test; *** P < 0.001

Article Snippet: Membranes were cultivated with primary antibodies EIF4G2 (1:1000, CST, #3468S) and GAPDH (1:4000, CST, #2118), followed by HRP-conjugated secondary antibody (Abcam, #ab205718).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Reverse Transcription, SDS Page, Binding Assay, Luciferase, Reporter Gene Assay, Transfection, Plasmid Preparation, Activity Assay

Journal: Cell Biology and Toxicology

Article Title: Effects of the LINC00641/miR-323a-3p/EIF4G2 axis on behaviors and brain monoamine neurotransmitters in chronic unpredictable mild stress mice

doi: 10.1007/s10565-025-10015-9

Figure Lengend Snippet: Inhibition of EIF4G2 hinders the effect of LINC00641 on depression-like behaviors of CUMS mice. A - D The expression level of EIF4G2 in the hippocampal tissues of mice in each group; E Results of sucrose preference experiments in mice treated with AAV-LINC00641 and AAV-shEF4G2; F - G Results of TST and FST in mice treated with AAV-LINC00641 and AAV-shEF4G2; H - J 5-HT, NE, and DA in brain tissues of mice treated with AAV-LINC00641 and AAV-shEF4G2; K Representative images of HE staining of hippocampal neurons in mice treated with AAV-LINC00641 and AAV-shEF4G2; L - M Apoptosis of hippocampal neurons in mice treated with AAV-LINC00641 and AAV-shEF4G2; n = 10 animals per group. The comparisons were made by Student’s t-test; ** P < 0.01, *** P < 0.001

Article Snippet: Membranes were cultivated with primary antibodies EIF4G2 (1:1000, CST, #3468S) and GAPDH (1:4000, CST, #2118), followed by HRP-conjugated secondary antibody (Abcam, #ab205718).

Techniques: Inhibition, Expressing, Staining